(13)C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled (13)C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-(13)C(2)]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-(13)C]glucose and [3-(13)C]glucose also outperformed the more commonly used [1-(13)C]glucose. [U-(13)C(5)]glutamine emerged as the preferred isotopic tracer for the analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of (13)C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture.